How to interpret thermal shift experiment's work for binding?

  • #1
udubson
2
1
Hello, PF I’m new here. Can someone please help me explain how to interpret thermal shift experiments work for binding? Apparently the data you receive from such experiment is a derivative dF/dT where F=fluorescence and T=temperature; and I’m very confused because isn’t the experiment supposed to help you determine when a protein begins to melt and denature? What does fluorescence have to do with this and does this mean you can only use proteins with GFP? I’m studying physics and proteomics is not my forte so apologies.
 
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  • #2
Do you understand qPCR? The assays’ method involves a dye that fluoresces during denaturation. The choice of dye is dependent on specific experiment and protein
 
  • #3
ProfuselyQuarky said:
Do you understand qPCR? The assays’ method involves a dye that fluoresces during denaturation. The choice of dye is dependent on specific experiment and protein
I would have to read more about it but I know it’s “real time PCR”. where does the derivative come to play exactly? And how does an inanimate dye fluoresce during denaturation?
 
  • #4
the dye interacts with protein residues that become exposed only when protein begins to unfold and only then does it fluoresce. So, more fluorescence corresponds with more unfolding. dF/dT is just the change in fluorescence with respect to temperature, so then you get very specific readings of what temp your protein begins to fold at.
 
  • Informative
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1. What is a thermal shift assay and how does it indicate binding?

A thermal shift assay, also known as differential scanning fluorimetry (DSF), measures the stability of proteins and their complexes under varying temperatures. The assay indicates binding by observing the change in the thermal stability of a protein upon the addition of a ligand or another protein. If the melting temperature (Tm) of the protein increases, it suggests that the binding of the ligand stabilizes the protein structure, indicating effective interaction.

2. How do you interpret an increase in melting temperature in a thermal shift assay?

An increase in the melting temperature (Tm) during a thermal shift assay typically indicates that the protein has become more thermally stable, which often results from effective binding with a ligand or another interacting molecule. This stabilization can be due to various interactions such as hydrophobic effects, hydrogen bonds, or changes in the protein's conformation that reduce its entropy.

3. What does a decrease in melting temperature suggest in a thermal shift assay?

A decrease in the melting temperature (Tm) in a thermal shift assay suggests that the interaction with a ligand or another protein makes the protein structure less stable. This could be due to the destabilization of the protein's native structure, potentially leading to partial unfolding or disruption of stabilizing interactions within the protein. It's important to consider the context and nature of the interaction to fully understand the implications of this observation.

4. How can you confirm that the observed thermal shift is due to specific binding?

To confirm that the observed thermal shift is due to specific binding, it's crucial to perform control experiments. These might include using different concentrations of the ligand to observe a dose-dependent change in Tm, conducting the assay with a known non-binder to rule out nonspecific effects, and repeating the experiment under different conditions or with different batches of protein. Additionally, complementary techniques such as isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR) can be used to confirm binding specificity and kinetics.

5. What are the limitations of thermal shift assays in studying protein-ligand interactions?

Thermal shift assays have several limitations. One major limitation is that they can only detect changes in thermal stability, not the nature of the binding interaction. They might not distinguish between different types of binding (e.g., allosteric vs. orthosteric) or between multiple binding sites. Furthermore, these assays require that the protein undergoes a significant change in thermal stability upon ligand binding, which may not always occur. Finally, the results can sometimes be influenced by factors unrelated to binding, such as changes in buffer conditions or the presence of impurities.

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